Fig. 3.

Hydrophobic gate. a Minimum distances of residue pairs forming the hydrophobic gate (magenta) in transition path at 100 °C with Na⁺ bound. b Concomitant changes of the two structural order parameters for domain motion (angle: blue, left scale; vertical displacement: red, right scale). c Structure of inward-open state, with inside access (orange) open and outside access (magenta) closed. Asp159 is shown in stick representation, and the bound Na⁺ as yellow sphere. Water molecules within 15 Å of the two carboxyl oxygen atoms of Asp159 are shown as spheres; other water molecules are shown as lines, and the protein backbone in gray and blue. d Structure of outward-open state. Arrows indicate opening and closing motions. e Transport assay of PaNhaP wild type and I163A/Y255A mutant reconstituted in liposomes. Transport was initiated by the dilution of (NH₄)₂SO₄-loaded proteoliposomes into reaction buffer. f Efflux of NH₃ from the liposomes creates a pH gradient that drives ²²Na uptake by PaNhaP. With 512.6 nmol min⁻¹ mg⁻¹, the ion exchange activity of the I163A/Y255A mutant is more than two times higher than wild type with 223.5 nmol min⁻¹ mg⁻¹. Error bars represent the standard error of the mean of ion exchange activity calculated from three independent measurements